Eukaryotic cells have evolved complex mechanisms to deal with environmental stresses. Signal transduction pathways are rapidly activated following exposure to DNA damaging agents and other cellular stresses, and these pathways affect processes such as gene transcription and cell cycle progression (Hartwell and Weinert (1989) Science 246:629–634; Hartwell and Kastan (1994) Science 266:1821–1828; Elledge (1996) Science 274:1664–1672). The protein encoded by the Ataxia-telangiectasia Mutated (ATM) locus, is a kinase critical for the initiation of signaling pathways following exposure of mammalian cells to ionizing radiation (IR) and to other agents that introduce double-strand breaks into cellular DNA (Kastan and Lim (2000) Mol. Cell Biol. 1:179–186; Shiloh and Kastan (2001) Adv. Cancer Res. 83:209–254). Cells from Ataxia-telangiectasia (A-T) patients typically lack detectable ATM protein, contain abnormalities in telomere morphology, and exhibit abnormal responses to IR, including increased cell death, increased chromosomal breakage, and cell cycle checkpoint defects (Shiloh (1997) Ann. Rev. Genet. 31:635–662). In addition, A-T patients exhibit progressive cerebellar ataxia, immune deficiencies, gonadal atrophy, oculocutaneous telangiectasias, radiation sensitivity, premature aging and increased risk of cancers, particularly lymphomas.
The ATM gene encodes a 370-kDa protein (Accession No. Q13315; SEQ ID NO:1) that belongs to the phosphoinositide 3-kinase (PI-3K) superfamily (Savitsky, et al. (1995) Science 268:1749–1753) which phosphorylates proteins rather than lipids (Banin, et al. (1998) Science 281:1674–1677; Canman, et al. (1998) Science 281:1677–1679). The 350 amino acid kinase domain at the C-terminus of this protein is the only segment of ATM with an assigned function. Exposure of cells to IR triggers ATM kinase activity and this function is required for arrests in G1, S, and G2 phases of the cell cycle (Shiloh and Kastan (2001) Adv. Cancer Res. 83:209–254). Several substrates of the ATM kinase participate in these IR-induced cell cycle arrests. For example, phosphorylation of p53, mdm2, and Chk2 govern the G1 checkpoint (Banin, et al. (1998) Science 281:1674–1677; Canman, et al. (1998) Science 281:1677–1679; Maya, et al. (2001) Genes Dev. 15:1067–1077; Matsuoka, et al. (2000) Proc. Natl. Acad. Sci. USA 97:10389–10394; Chehab, et al. (2000) Genes Dev. 14:278–288); Nbs1, Brca1, FancD2, and SMC1 participate in the transient IR-induced S-phase arrest (Lim, et al. (2000) Nature 404:613–617; Wu, et al. (2000) Nature 405:477–482; Zhou, et al. (2000) J. Biol. Chem. 275:10342–10348; Taniguchi, et al. (2002) Cell 109:459–472; Kim, et al. (2002) Genes Dev. 16:560–570; Yazdi, et al. (2002) Genes Dev. 16:571–582; Xu, et al. (2002) Cancer Res. 62:4588–4591); and Brca1 and hRad17 have been implicated in the G2/ M checkpoint (Xu, et al. (2001) Mol. Cell. Biol. 21:3445–3450; Bao, et al. (2001) Nature 411:969–974).
The mechanisms by which eukaryotic cells sense DNA strand breaks is unknown, but the rapid induction of ATM kinase activity following IR indicates that it acts at an early stage of signal transduction in mammalian cells (Banin, et al. (1998) Science 281:1674–1677; Canman, et al. (1998) Science 281:1677–1679). Transfected ATM is a phosphoprotein that incorporates more phosphate after IR treatment of cells (Lim, et al. (2000) Nature 404:613–617), suggesting that ATM kinase is itself activated by post-translational modification.
Inhibiting ATM for the treatment of neoplasms, particularly cancers associated with decreased p53 function, has been suggested (Morgan, et al. (1997) Mol. Cell Biol. 17:2020–2029; Hartwell and Kastan (1994) Science 266:1821–1828; Kastan (1995) New Eng. J. Med. 333:662–663; WO 98/56391). WO 98/56391 further provides genetically manipulated knock-out mice as a model for testing ATM inhibitors and suggests the use of an inhibitory antibody to ATM, a dominant-negative fragment of ATM or an ATM antisense strategy to inhibit ATM.
U.S. Pat. No. 6,387,640 discloses the use of an ATM kinase substrate recognition sequence in an assay system to screen for compounds that modulate ATM-mediated phosphorylation. The substrate recognition sequence provided comprises Xaa1-Xaa-Xaa2-Ser-Gln-Xaa-Xaa (SEQ ID NO:2) wherein Xaa1 is a hydrophobic amino acid, Xaa2 is a hydrophobic amino acid or aspartic acid, and Xaa is any amino acid.
U.S. Pat. No. 6,348,311 further discloses a method of identifying an inhibitor of ATM-mediated kinase activity by determining the extent of cell survival after HTLV infection.